Identification of a serum and urine extracellular vesicle signature predicting renal outcome after kidney transplant

Background A long-standing effort is dedicated towards the identification of biomarkers allowing the prediction of graft outcome after kidney transplant. Extracellular vesicles (EVs) circulating in body fluids represent an attractive candidate, as their cargo mirrors the originating cell and its pathophysiological status. The aim of the study was to investigate EV surface antigens as potential predictors of renal outcome after kidney transplant. Methods We characterized 37 surface antigens by flow cytometry, in serum and urine EVs from 58 patients who were evaluated before, and at 10-14 days, 3 months and 1 year after transplant, for a total of 426 analyzed samples. The outcome was defined according to estimated glomerular filtration rate (eGFR) at 1 year. Results Endothelial cells and platelets markers (CD31, CD41b, CD42a and CD62P) in serum EVs were higher at baseline in patients with persistent kidney dysfunction at 1 year, and progressively decreased after kidney transplant. Conversely, mesenchymal progenitor cell marker (CD1c, CD105, CD133, SSEEA-4) in urine EVs progressively increased after transplant in patients displaying renal recovery at follow-up. These markers correlated with eGFR, creatinine and proteinuria, associated with patient outcome at univariate analysis and were able to predict patient outcome at receiver operating characteristics curves analysis. A specific EV molecular signature obtained by supervised learning correctly classified patients according to 1-year renal outcome. Conclusions An EV-based signature, reflecting the cardiovascular profile of the recipient, and the repairing/regenerative features of the graft, could be introduced as a non-invasive tool for a tailored management of follow-up of patients undergoing kidney transplant

Characterisation of viral genomes by MALDI-TOF mass spectrometry in serum and urinary nanovescicles isolated from patients undergoing renal transplantation

Gli esosomi, vescicole nanometriche rilasciate dalla maggior parte delle cellule presenti nei vari fluidi biologici, possono trasferire il loro carico di proteine e acidi nucleici alle cellule riceventi. Scopo del presente studio è stato quello di valutare la presenza di genomi virali,appartenenti alle famiglie dei PyVs e degli HPV, associati a NV (NV) in 72 pazienti sottoposti a trapianto renale, in campioni di sangue e urina L’individuazione dei genomi virali è stata effettuata mediante spettrometria di massa MALDI- TOF. consentendo la contemporanea rilevazione dei 18 genomi virali per i PyV e dei 16 HPV per ciascun campione sottoposto ad analisi. Nei soggetti reclutati, donatori e riceventi, è stato effettuato inizialmente l’isolamento delle NV in campioni di siero e urina utilizzando una metodica di precipitazione basata sul polietilenglicole (PEG). La determinazione del numero e delle dimensioni delle NV isolate è stata effettuata mediante NTA. La conseguente determinazione degli epitopi esosomiali e nano vescicolari supercifiali è stata condotta mediante metodica MACSplex. L’arricchimento nelle NV urinarie della frazione esoosmiale è emerso grazie all’elevata presenza dell’antigene CD9 sulla superficie delle NV stesse. Inoltre, l’utilizzo della digestione enzimatica mediata da DNAsi I ha permesso di testare quanto potesse essere aspecifica la rilevazione dei genomi virali identificati con la spettrometria di massa, qualora si palesassero delle copurificazioni inattese. L’analisi dei genomi virali è stata così condotta in parallelo testando i medesimi campioni trattati con DNAsi I e non. I risultati ottenuti sulla corte dei soggetti donatori e dei pazienti trapiantati hanno messo in luce, delle percentuali di positività spiccate per il genoma virale del BKV (60%) e del JCV (20%) nelle NV sieriche ma soprattutto in quelle urinarie (50% entrambi).Advances in surgical techniques and the evolution of immunosuppressive therapy have undoubtedly improved survival rates, but post-transplant infections continue to cause morbidity and mortality in these patients. Exosomes, nanometric vesicles released by most of the cells present in the various biological fluids, can transfer their load of proteins and nucleic acids to the receiving cells. The aim of this study was to evaluate the presence of viral genomes, belonging to the families of PyVs and HPVs, associated with nanovescicles, in 72 patients undergoing renal transplantation, in samples of blood and urine. The identification of viral genomes was carried out through the development of a high-throughput method based on mass spectrometry MALDI- TOF. The method allowed the simultaneous detection of 18 viral genomes for PyV and 16 HPV for each sample tested. The isolation of the NV was initially carried out using a precipitation method based on PEG. The number and size of the isolated NV was determined by NTAs. The determination of exosomal epitopes was performed by the MACSplex method. The enrichment of the exosomial fraction in the urinary NVs was revealed by the high presence of the CD9 antigen on the surface of the NVs themselves. Moreover, the use of enzymatic digestion allowed to test how unspecific the detection of viral genomes identified with mass spectrometry could be, in case of unexpected copurifications. The results obtained on the transplanted patients have shown marked positive percentages for the viral genome of BKV (60%) and JCV (20%) in serum NV but especially in urinary ones (50% both). In conclusion, the investigation carried out in this project has led to the identification of the combination of isolation by immunomagnetic separation and enzyme treatment with DNAase I as the best method for the determination of viral genomes contained within exosomes (.i.e. SV12)

Modifiers of Autosomal Dominant Polycystic Kidney Disease Severity: The Role of <i>PKD1</i> Hypomorphic Alleles

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of kidney failure in adult life. Rarely, ADPKD can be diagnosed in utero or in infancy, and the genetic mechanism underlying such severe presentation has been shown to be related to reduced gene dosage. Biallelic PKD1 variants are often identified in early onset ADPKD, with one main pathogenic variant and a modifier hypomorphic variant showing an in trans configuration. We describe two unrelated individuals with early onset cystic kidney disease and unaffected parents, where a combination of next-generation sequencing of cystic genes including PKHD1, HNF1B and PKD1 allowed the identification of biallelic PKD1 variants. Furthermore, we review the medical literature in order to report likely PKD1 hypomorphic variants reported to date and estimate a minimal allele frequency of 1/130 for this category of variants taken as a group. This figure could help to orient genetic counseling, although the interpretation and the real clinical impact of rare PKD1 missense variants, especially if previously unreported, remain challenging

Methylenetetrahydrofolate reductase, MTHFR, polymorphisms and predisposition to different multifactorial disorders

Gene polymorphisms involved in homocysteine-methionine pathway result in hyperhomocysteinemia, a predisposing condition to several diseases. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate and homocysteine metabolism. The two known functional polymorphisms of MTHFR gene, 677C>T and 1298A>C have been implicated in a variety of multifactorial diseases: cardio-cerebrovascular and neurodegenerative disorders, autoimmune diseases, birth defects, diabetes, neuropsychiatric disorders, cancer and renal disease. C667T, and to a lesser extent A1298C polymorphisms, have been also reported to have a pharmacogenetic role in predicting drug toxicity in cancer and rheumatoid arthritis treatment. We review here the principal effects of the MTHFR gene variations in different clinical conditions

DNA VIRAL INFECTIONS ASSAY IN KIDNEY TRANSPLANT RECIPIENTS BY A NEW HIGH THROUGHPUT MASS SPECTROMETRY METHOD

Introduction: Infections represent a major complication after renal transplant with an important impact on allograft survival and outcome. Polyomaviruses (PyVs), a group of small and circular dsDNA viruses, mediate a broad spectrum of diseases in immune-compromised patients. NF-jB (nuclear factor kappalight-chain-enhancer of activated B cells) is a key regulator of immune and inflammatory responses and the -94ins/delATTG (rs28362491) polymorphism in the gene promoter has been widely investigated for clinical associations. To date, rs28362491 has been shown to influence the susceptibility to systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease and recently renal transplant rejection. Materials and Methods: We developed a high throughput mass spectrometry (MS)-based method to detect rs28362491 and 18 Py vs. types. Primer pairs of MS assay were designed within the specific large T antigen genes. Viral and human DNAs were extracted from blood samples of 43 kidney transplant recipients, before and after transplantation. Results: We analysed the correlation among Py vs. infections, rs28362491 genotype and post-transplant follow up. Five out of the 18 viral types tested were found in the specimens analysed: BKV, JCV, Merkel cell PyV, Human PyV6 and SV12. In our cohort, 14 patients showed SV12 infection: 10 cases were -94ins/-94ins, 4 were -94ins/-94del. All the patients with the NF-kB-94del/- 94del genotype were characterized by the absence of SV12 strain. No correlation between genotype and viral infection was observed for the other viral types. Conclusions: Our MS assay improved the Py vs. typing and allowed to drive towards the identification of novel biomarkers for the infective management of transplanted patients. The genetic background might modulate the viral infection susceptibility in renal transplant recipients

Pleiotropic genes in psychiatry:Calcium channels and the stress-related FKBP5 gene in antidepressant resistance

A candidate gene and a genome-wide approach were combined to study the pharmacogenetics of antidepressant response and resistance. Investigated genes were selected on the basis of pleiotropic effect across psychiatric phenotypes in previous genome-wide association studies and involvement in antidepressant response. Three samples with major depressive disorder (total = 671) were genotyped for 44 SNPs in 8 candidate genes (CACNA1C, CACNB2, ANK3, GRM7, TCF4, ITIH3, SYNE1, FKBP5). Phenotypes were response/remission after 4 weeks of treatment and treatment-resistant depression (TRD). Genome-wide data from STAR*D were used to replicate findings for response/remission (n = 1409) and TRD (n = 620). Pathways including the most promising candidate genes were investigated in STAR*D for involvement in TRD. FKBP5 polymorphisms showed replicated but nominal associations with response, remission or TRD. CACNA1C rs1006737 and rs10848635 were the only polymorphisms that survived multiple-testing correction. In STAR*D the best pathway associated with TRD included CACNA1C (GO:0006942, permutated p = 0.15). Machine learning models showed that independent SNPs in this pathway predicted TRD with a mean sensitivity of 0.83 and specificity of 0.56 after 10-fold cross validation repeated 100 times. FKBP5 polymorphisms appear good candidates for inclusion in antidepressant pharmacogenetic tests. Pathways including the CACNA1C gene may be involved in TRD and they may provide the base for developing multi-marker predictors of TRD.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Genetic Variants Within Molecular Targets of Antipsychotic Treatment: Effects on Treatment Response, Schizophrenia Risk, and Psychopathological Features

Schizophrenia (SCZ) is a common and severe mental disorder. Genetic factors likely play a role in its pathophysiology as well as in treatment response. In the present study, we investigated the effects of several single nucleotide polymorphisms (SNPs) within 9 genes involved with antipsychotic (AP) mechanisms of action. Two independent samples were recruited. The Korean sample included 176 subjects diagnosed with SCZ and 326 healthy controls, while the Italian sample included 83 subjects and 194 controls. AP response as measured by the positive and negative syndrome scale (PANSS) was the primary outcome, while the secondary outcome was the SCZ risk. Exploratory analyses were performed on (1) symptom clusters response (as measured by PANSS subscales); (2) age of onset; (3) family history; and (4) suicide history. Associations evidenced in the primary analyses did not survive to the FDR correction. Concerning SCZ risk, we partially confirmed the associations among COMT and MAPK1 genetic variants and SCZ. Finally, our exploratory analysis suggested that CHRNA7 and HTR2A genes may modulate both positive and negative symptoms responses, while PLA2G4A and SIGMAR1 may modulate respectively positive and negative symptoms responses. Moreover, GSK3B, HTR2A, PLA2G4A, and S100B variants may determine an anticipation of SCZ age of onset. Our results did not support a primary role for the genes investigated in AP response as a whole. However, our exploratory findings suggested that these genes may be involved in symptom clusters response